A型口蹄疫VP1基因的原核表达及纯化  

Prokaryotic expression and purification of the VP1 gene from Foot- and- mouth disease virus( FMDV) type A

作　　者：刘清源[1,2] 向华[1] 黄元[1] 陈晶[1] 向蓉[1] 郑海学[3] 刘宇[2] 

机构地区：[1]广东省农业科学院动物卫生研究所/广东省兽医公共卫生公共实验室,广州510640 [2]华南农业大学兽医学院,广州510642 [3]中国农业科学院兰州兽医研究所/家畜疫病病原微生物国家重点实验室,兰州730046

出　　处：《黑龙江畜牧兽医》2016年第5期173-176,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：科技部农业科技成果转化基金项目(2013GBZE000352);广东省科技厅重点实验室建设支撑项目(2012A061100006);广东省农业科学院院长基金项目(201530);广东省主体机构创新能力建设专项(2011)

摘　　要：为了研究A型口蹄疫病毒(FMDV)VP1蛋白的免疫原性,试验采用PCR方法从口蹄疫阳性质粒p MD-18-P1中扩增得到目的基因VP1,与原核表达载体p ET-28a连接,构建重组质粒p ET-28a-VP1,转化大肠杆菌Rosetta(DE3)感受态细胞,筛选的阳性菌落用IPTG诱导表达,收集菌液进行SDS-PAGE电泳,通过Western-blot分析蛋白的免疫原性,并用镍亲和树脂纯化鉴定后的重组蛋白。结果表明:重组质粒p ET-28a-VP1经PCR及酶切鉴定证明构建正确;重组质粒能够在大肠杆菌中大量表达,表达产物的分子质量大小约为26 ku,且能被口蹄疫阳性血清识别;镍亲和树脂纯化的蛋白条带单一。说明该蛋白具有免疫学活性,且纯度较高。To study the immunogenicity of VPI protein of Foot - and - mouth disease virus （FMDV） type A, a target gene VPI was amplified from the plasmid pMD- 18 -P1 of foot -and -mouth disease （FMD） using PCR assay. The target gene VP1 was linked with prokaryotic expression vector pET - 28a vector to construct a reeombined plasmid pET - 28a - VP1, and then the recombined plasmid was transformed into Escherichia coli Rosetta （ DE3 ） competent cells. The positive colonies were screened, and then the expression of protein was induced by IPTG. The bacterial liquids were collected for SDS -PAGE electmphoresis, and then the immunogenicity of protein was analyzed by Western -blot. The identified recombinant protein was purified with Ni - NTA resin. The result showed that the recombinant plasmid pET - 28a - VP1 was constructed correctly, which was proved by PCR and identification of restriction enzyme digestion; the recombinant plasmid could be expressed abundantly in Escherichia coli, and the molecular mass of expressed product was approximately 26 ku, which could be recognized by FMD - positive sera; the bands of the protein purified by Ni - NTA resin were single. The results indicate that the protein has immunological activity with higher purity.

关 键 词：口蹄疫病毒(FMDV) VP1基因 克隆表达 鉴定 纯化 

分 类 号：S855.3[农业科学—临床兽医学] Q786[农业科学—兽医学]